These projected experiments are designed to extend our knowledge of the DNA replication process 1) by combining biochemical and genetic studies to elucidate the molecular biology of the initiation process and 2) by utilizing two separate series of mutants to define the roles played by the products of the mutated genes in DNA replication. We plan to exploit our recently developed in vitro initiation assay to purify the gene products active in the initiation process. The ultimate goal of this approach is the development of a purified in vitro chromosome initiation system that can be used to study the molecular mechanism of this regulatory step in DNA replication. The regulation of the dnaA gene will be examined by placing the lac genes under the dnaA control mechanism. The production of the easily assayable enzyme, B-galactosidase, will, thus, reflect the regulation of the dnaA gene. Several dna mutations are suppressed amber codons. By substituting the appropriate temperature-sensitive suppressor for the originally present supE, the products of these genes will then be made dependent on the condition of the suppressor. The abolition of suppressor action, while allowing continued cell growth, will result in titration of the particular dna gene product. The effect of the lowered concentration of gene product on the DNA synthetic mechanism can then be examined. The genes involved in this set of experiments are dnaA, dnaB, and gyrB. Finally, a series of RNaseH mutants have been isolated, and they will be studied biochemically and genetically to determine if this enzyme functions in normal DNA replication.